Thyroid hormone differentially augments biliary sterol secretion in the rat. I. The isolated-perfused liver model.

Thyroid hormone lowers serum cholesterol and alters sterol metabolic processes. This laboratory has previously reported increased biliary lipid secretion as an early effect of triiodothyronine (T3) in the rat. To evaluate whether the bile lipid action of T3 is a primary or secondary effect, the isolated-perfused rat liver model was used. Red blood cells in lipid-free buffer were used to perfuse livers of euthyroid and methimazole-hypothyroid rats, as well as hypothyroid rats given T3 at intervals before perfusion. Bile flow was maintained by taurocholate perfusion. Hypothyroid rats had elevated pre-perfusion serum cholesterol compared to euthyroid (107 +/- 4 vs. 65 +/- 2 mg/dl) and decreased biliary cholesterol (0.016 +/- 0.001 vs. 0.031 +/- 0.004 mumol/g liver/h) secretion. Serum cholesterol decreased to euthyroid levels by 18 h after T3, an effect that was prevented by bile duct ligation. Bile cholesterol secretion doubled by 18 h, and reached levels twice euthyroid by 42 h, while phospholipid secretion doubled to levels just above euthyroid. The fourfold increase in biliary cholesterol secretion occurred with lipid-free perfusion and unchanging bile acid uptake or output. It occurred without a fall in hepatic lipoprotein cholesterol secretion. Blockade of cholesterol synthesis with lovastatin failed to alter T3-augmented bile cholesterol secretion. We conclude that T3 induces biliary cholesterol secretion concomitantly with the fall in serum cholesterol. This augmented biliary secretion did not appear to depend upon lipoprotein uptake, increased bile acid transport, or cholesterol synthesis. It did not occur at the expense of hepatic lipoprotein secretion. Facilitated biliary lipid secretion may be a primary effect of T3.     

Thyroid hormone differentially augments biliary sterol secretion in the rat. II. The chronic bile fistula model.

To further define thyroid hormone effects on bile acid synthesis and biliary lipid secretion, studies were done in chronic bile fistula rats. Euthyroid and methimazole-hypothyroid rats, with and without triiodothyronine (T3) injection, had total bile diversion for timed bile collections. With interrupted enterohepatic circulation, cholesterol absorption is negligible and bile acid secretion equals bile acid synthesis rate. Hypothyroid rats had diminished levels of bile acid synthesis and biliary secretion of cholesterol and phospholipid. Single dose T3 injection produced a 13-fold increase in bile cholesterol secretion and a 3-fold increase in phospholipid secretion, both initiated 12 h after T3. Bile acid synthesis increased by 50%, but the increase did not begin until 24 h after T3. Neither hypothyroidism nor T3 treatment abolished diurnal rhythms of bile acid synthesis and biliary lipid secretion. Inhibition of cholesterol synthesis with lovastatin resulted in a persistent 33% decrease in bile acid synthesis in euthyroid and hypothyroid rats, while bile cholesterol secretion only transiently decreased. Inhibition of cholesterol synthesis did not alter T3-induced bile cholesterol secretion, with a 10-fold increase seen. However, bile acid synthesis was not stimulated by T3 in the presence of lovastatin. We conclude that facilitated bile acid synthesis and biliary cholesterol secretion are early effects of T3 and may account for the hypocholesterolemia of T3. Cholesterol synthesis does not appear to be required for the T3-induced bile cholesterol secretion.     

Hepatic uptake and metabolism of free cholesterol from different lipoprotein classes. An in vivo study in the rat

Hepatic metabolism of [14C]cholesterol, vehiculated by LDL, HDL2 and HDL3 lipoprotein particles, has been studied in rats with a permanent biliary drainage. The lipoprotein fractions were infused individually by a jugular vein catheter and bile was collected for 180 min after the administration. At the end of this period, the animals were killed and the blood and livers were collected. The free cholesterol of the HDL2 fraction was secreted into bile, mainly as bile salt, preferentially to that associated with HDL3 and LDL fractions (11.7% vs. 2.3% and 0.3%, respectively). The free cholesterol of the HDL3 fraction, on the other hand, was taken up by liver more quickly and in a higher proportion than that associated with other lipoprotein fractions. The label incorporation in this lipoprotein fraction was secreted earlier and not transformed into bile. The contribution of LDL-vehiculated free cholesterol to bile secretion was small and the hepatic uptake amounted to no more than 12% of the injected label.     

Effect of bean intake on biliary lipid secretion and on hepatic cholesterol metabolism in the rat

We studied the effect of a bean diet on biliary lipid secretion, serum cholesterol concentration, and hepatic cholesterol metabolism in the rat. Rats fed a bean diet for 10-12 days had increased biliary cholesterol output and molar percentage by 300% and 200%, respectively, compared to rats fed an isocaloric and isoprotein casein diet. Biliary phospholipid output increased 180%. Bile flow and biliary bile salt output remained in the normal range. Total serum and VLDL cholesterol concentration significantly decreased 27% and 50%, respectively, in the rats fed the bean diet. Hepatic cholesterogenesis was increased 170% in the bean-fed animals. The relative contribution of newly synthesized hepatic cholesterol to total biliary cholesterol increased 200%, and that of endogenous origin only 50%. These results suggested that newly synthesized hepatic cholesterol was preferentially channelled to the biliary cholesterol secretory pathway in bean-fed rats. Although hepatic cholesteryl ester concentration increased 240%, the incorporation of [14C]oleate into hepatic cholesteryl esters was significantly decreased by 30% in isolated hepatocytes of bean-fed animals. These results were consistent with the possibility that the availability of hepatic free cholesterol for biliary secretion was increased in the bean-fed animals. This study demonstrates that bean intake has a profound effect on the metabolic channelling and compartmentalization of hepatic cholesterol, resulting in a significant decrease in total serum and very low density lipoprotein cholesterol concentrations and a high biliary cholesterol output.     

Processing of cholesteryl ester from low-density lipoproteins in the rat. Hepatic metabolism and biliary secretion after uptake by different hepatic cell types.

Biliary secretion of the cholesteryl ester moiety of (modified) low-density lipoprotein (LDL) was examined under various experimental conditions in the rat. Human LDL or acetylated LDL (acetyl-LDL), radiolabelled with [3H]cholesteryl oleate, was administered intravenously to unanesthetized rats equipped with permanent catheters in the bile duct, duodenum and heart. LDL was cleared relatively slowly from plasma, mainly by Kupffer cells. At 3 h after injection, only 0.9% of the radioactivity was found in bile; after 12 h this value was 4.5%. Uptake of LDL by hepatocytes was stimulated by treatment of the rats with 17 alpha-ethinyloestradiol (EE; 5 mg/kg for 3 successive days); this resulted in a more rapid secretion of radioactivity into bile, 3.9% and 12.4% after 3 h and 12 h respectively. The extremely rapid uptake of acetyl-LDL via the scavenger pathway, mainly by endothelial cells, resulted in the secretion of only 2.1% of its 3H label into bile within 3 h, and 9.5% within 12 h. Radioactivity in bile was predominantly in the form of bile acids; only a small part was secreted as free cholesterol. However, the specific radioactivity of biliary cholesterol was higher than that of bile acids in all three experimental conditions. EE-treated animals did not form cholic acid from [3H]cholesteryl oleate, which was a major product of the cholesteryl oleate from LDL and acetyl-LDL in untreated rats, but formed predominantly very polar bile acids, i.e. muricholic acids. It is concluded that uptake of human LDL or acetyl-LDL by the liver of untreated rats is not efficiently coupled to biliary secretion of cholesterol (bile acids). This might be due to the anatomical localization of their principal uptake sites, the Kupffer cells and the endothelial cells respectively. Induction of LDL uptake by hepatocytes by EE treatment warrants a more efficient disposition of cholesterol from the body via bile.     

Mechanism and kinetic characteristics of the uncoupling by plant steroids of biliary cholesterol from bile salt output

In male Wistar rats fed diets containing different plant steroids, including sitosterols, diosgenin, digitonin and saponin from gypsophila, biliary cholesterol secretion significantly increased 50% to 300%, whereas biliary bile salt and phospholipid showed minor changes. Both cholesterol and phospholipid outputs were coupled to biliary bile salt output in a curvi-linear relationship which could be fitted by rectangular hyperbolae, in the animals fed with different plant steroids. The theoretical maximal biliary cholesterol output significantly increased by 200% in sitosterol-fed rats and 500% in diosgenin-fed animals. No changes were found in the kinetic characteristics of biliary phospholipid outputs. Adding 2% cholesterol to the diosgenin diet abolished the increment of biliary cholesterol output induced by the plant steroid. The intraperitoneal injection of 45 mumol/kg body wt per day (3 days) diosgenin, a C27-sapogenin, and 65 mumol/kg body wt. per day (3 days) tomatidin, a C27-alkaloid, incorporated in phosphatidylcholine-taurocholate liposomes significantly increased biliary cholesterol output by 70%. These experiments indicated that the plant steroid-induced biliary cholesterol output was independent of the inputs of cholesterol from the diet and from hepatic cholesterogenesis modified by the plant steroid. It was apparent that the profound changes of biliary cholesterol secretion were the consequence of direct effects of the steroids on the intrahepatocytic regulatory mechanisms of biliary cholesterol secretion. This novel effect appears to be a universal characteristic of plant steroids, since it can be elicited by sitosterols, C27-sapogenins, C27-alkaloids, and saponins of the cholanic and beta-amirinic group.     

Hepatic processing of the cholesteryl ester from low density lipoprotein in the rat

Human low density lipoprotein (LDL), radiolabeled in the cholesteryl ester moiety, was injected into estrogen-treated and -untreated rats. The hepatic and extrahepatic distribution and biliary secretion of [3H]cholesteryl esters were determined at various times after injection. In order to follow the intrahepatic metabolism of the cholesteryl esters of LDL in vivo, the liver was subfractioned into parenchymal and Kupffer cells by a low temperature cell isolation procedure. In control rats, the LDL cholesteryl esters were mainly taken up by the Kupffer cells. After uptake, the [3H]cholesteryl esters are rapidly hydrolyzed, followed by release of [3H]cholesterol from the cells to other sites in the body. Up to 24 h after injection of LDL, only 9% of the radioactivity appeared in the bile, whereas after 72 h, this value was 30%. Hepatic and especially the parenchymal cell uptake of [3H]cholesteryl esters from LDL was strongly increased upon 17 alpha-ethinylestradiol treatment (3 days, 5 mg/kg). After rapid hydrolysis of the esters, [3H]cholesterol was both secreted into bile (28% of the injected dose in the first 24 h) as well as stored inside the cells as re-esterified cholesterol ester. It is concluded that uptake of human LDL by the liver in untreated rats is not efficiently coupled to biliary secretion of cholesterol (derivatives), which might be due to the anatomical localization of the principal uptake site, the Kupffer cells. In contrast, uptake of LDL cholesterol ester by liver hepatocytes is tightly coupled to bile excretion. The Kupffer cell uptake of LDL might be necessary in order to convert LDL cholesterol (esters) into a less toxic form. This activity can be functional in animals with low receptor activity on hepatocytes, as observed in untreated rats, or after diet-induced down-regulation of hepatocyte LDL receptors in other animals.     

Biliary lipid secretion in patients with heterozygous familial hypercholesterolemia and combined hyperlipidemia. Influence of bezafibrate and fenofibrate

Serum and biliary lipid metabolism were examined in 13 patients with different types of hyperlipoproteinemia before and after 4 weeks of treatment with either bezafibrate or fenofibrate. In patients with heterozygous familial hypercholesterolemia (FH), bezafibrate (n = 5) and fenofibrate (n = 7) produced a similar significant reduction of total cholesterol, LDL-cholesterol, and triglycerides by 21, 23, and 32%, respectively. In patients with familial combined hyperlipidemia (CHL), only triglycerides decreased markedly. Biliary lipid secretion rates in patients with heterozygous FH were not different from those of young male volunteers, indicating that a reduction of hepatic LDL receptors did not affect hepatic elimination of cholesterol or bile acids. Biliary cholesterol secretion increased significantly from 57 to 75 mg/hr during bezafibrate therapy (n = 8) and from 62 to 71 mg/hr during fenofibrate therapy (n = 9). No consistent change in bile acid or phospholipid secretion was observed. The elevated output of biliary cholesterol increased cholesterol saturation significantly from 147 to 185% and from 152 to 173% during administration of bezafibrate and fenofibrate, respectively. The present study indicates that treatment with bezafibrate or fenofibrate is effective in lowering LDL cholesterol in patients with heterozygous FH, but both drugs increase cholesterol saturation of bile, which might enhance the risk of cholesterol gallstone formation.     

Effects of dietary soybean lecithin on plasma lipid transport and hepatic cholesterol metabolism in rats

Dietary lecithin can stimulate bile formation and biliary lipid secretion, particularly cholesterol output in bile. Studies also suggested that the lecithin-rich diet might modify hepatic cholesterol homeostasis and lipoprotein metabolism. Therefore, we examined hepatic activities of 3-hydroxy-3 methylglutaryl coenzyme A reductase "HMG -CoA reductase", cholesterol 7 alpha-hydroxylase and acyl-CoA: cholesterol acyltransferase "ACAT" as well as plasma lipids and lipoprotein composition in rats fed diets enriched with 20% of soybean lecithin during 14 days. We also evaluated the content of hepatic canalicular membrane proteins involved in lipid transport to the bile (all P-glycoproteins as detected by the C 219 antibody and the sister of P-glycoprotein "spgp" or bile acid export pump) by Western blotting. As predicted, lecithin diet modified hepatic cholesterol homeostasis. The activity of hepatic HMG-CoA reductase and cholesterol 7 alpha-hydroxylase was enhanced by 30 and 12% respectively, while microsomal ACAT activity showed a dramatic decrease of 75%. As previously reported from ACAT inhibition, the plasma level and size of very low-density lipoprotein (VLDL) were significantly decreased and bile acid pool size and biliary lipid output were significantly increased. The canalicular membrane content of lipid transporters was not significantly affected by dietary lecithin. The current data on inhibition of ACAT activity and related metabolic effects by lecithin mimic the previously reported effects following drug-induced inhibition of ACAT activity, suggesting potential beneficial effects of dietary lecithin supplementation in vascular disease.     

Hepatic cholesterol and bile acid metabolism and intestinal cholesterol absorption in scavenger receptor class B type I-deficient mice

The scavenger receptor class B type I (SR-BI), which is expressed in the liver and intestine, plays a critical role in cholesterol metabolism in rodents. While hepatic SR-BI expression controls high density lipoprotein (HDL) cholesterol metabolism, intestinal SR-BI has been proposed to facilitate cholesterol absorption. To evaluate further the relevance of SR-BI in the enterohepatic circulation of cholesterol and bile salts, we studied biliary lipid secretion, hepatic sterol content and synthesis, bile acid metabolism, fecal neutral sterol excretion, and intestinal cholesterol absorption in SR-BI knockout mice. SR-BI deficiency selectively impaired biliary cholesterol secretion, without concomitant changes in either biliary bile acid or phospholipid secretion. Hepatic total and unesterified cholesterol contents were slightly increased in SR-BI-deficient mice, while sterol synthesis was not significantly changed. Bile acid pool size and composition, as well as fecal bile acid excretion, were not altered in SR-BI knockout mice. Intestinal cholesterol absorption was somewhat increased and fecal sterol excretion was slightly decreased in SR-BI knockout mice relative to controls. These findings establish the critical role of hepatic SR-BI expression in selectively controlling the utilization of HDL cholesterol for biliary secretion. In contrast, SR-BI expression is not essential for intestinal cholesterol absorption.     

Disruption of the sterol carrier protein 2 gene in mice impairs biliary lipid and hepatic cholesterol metabolism.

Hepatic up-regulation of sterol carrier protein 2 (Scp2) in mice promotes hypersecretion of cholesterol into bile and gallstone formation in response to a lithogenic diet. We hypothesized that Scp2 deficiency may alter biliary lipid secretion and hepatic cholesterol metabolism. Male gallstone-susceptible C57BL/6 and C57BL/6(Scp2(-/-)) knockout mice were fed a standard chow or lithogenic diet. Hepatic biles were collected to determine biliary lipid secretion rates, bile flow, and bile salt pool size. Plasma lipoprotein distribution was investigated, and gene expression of cytosolic lipid-binding proteins, lipoprotein receptors, hepatic regulatory enzymes, and intestinal cholesterol absorption was measured. Compared with chow-fed wild-type animals, C57BL/6(Scp2(-/-)) mice had higher bile flow and lower bile salt secretion rates, decreased hepatic apolipoprotein expression, increased hepatic cholesterol synthesis, and up-regulation of liver fatty acid-binding protein. In addition, the bile salt pool size was reduced and intestinal cholesterol absorption was unaltered in C57BL/6(Scp2(-/-)) mice. When C57BL/6(Scp2(-/-)) mice were challenged with a lithogenic diet, a smaller increase of hepatic free cholesterol failed to suppress cholesterol synthesis and biliary cholesterol secretion increased to a much smaller extent than phospholipid and bile salt secretion. Scp2 deficiency did not prevent gallstone formation and may be compensated in part by hepatic up-regulation of liver fatty acid-binding protein. These results support a role of Scp2 in hepatic cholesterol metabolism, biliary lipid secretion, and intracellular cholesterol distribution.     

Differential stimulation of S-adenosylmethionine decarboxylase by difluoromethylornithine in the rat colon and small intestine.

The effects of sodium cyclobutyrate, a synthetic hydrocholeretic drug, on biliary lipid secretion and on the biliary outputs of several plasma-membrane enzymes were investigated in anaesthetized rats. Administration of a single oral dose of cyclobutyrol (0.72 mmol/kg body wt.) reduced biliary concentration and output of cholesterol and phospholipid. However, bile acid secretion was not significantly modified. This uncoupling effect of lipid secretion remained even when the choleretic response to the drug had ceased. It additionally led to a statistically significant decrease in the cholesterol/bile acid and phospholipid/bile acid molar ratios and in the lithogenic index of the bile. The biliary outputs of the plasma-membrane enzymes alkaline phosphatase and gamma-glutamyltransferase were markedly reduced by the drug. When cyclobutyrol was administered to rats which had been previously fed with a high-cholesterol diet, the effects of cyclobutyrol persisted, but were less marked. Our results demonstrate that the bile acid-independent choleresis induced by cyclobutyrol (related to its pharmacokinetic effect) is accompanied by a pharmacodynamic action that selectively reduces the secretion of biliary lipids. This is due to an uncoupling of the secretion of cholesterol and phospholipids from that of bile acids. Possible explanations for the biliary response to cyclobutyrol are discussed.     

Abnormal hepatobiliary and circulating lipid metabolism in the Long-Evans Cinnamon rat model of Wilson's disease

Long-Evans Cinnamon (LEC) rats exhibit a genetic defect in Atp7b gene, which is homologous to the human Wilson's disease gene, resulting in an inability to mobilize copper from the liver. This study was undertaken to gain insight into the relationship between liver copper accumulation and plasma lipid profile, circulating lipoprotein composition, hepatic sterol metabolism and biliary lipid secretion rates in 12-week-old LEC rats compared to control Long-Evans rats. Concomitant with hepatic copper deposition, LEC rats displayed increased content of triglycerides (TGs), free cholesterol (FC) and cholesteryl ester (CE) in the liver. Hepatic concentrations of malondialdehyde (MDA), an index of lipid peroxidation were also significantly elevated in LEC rats (50%). This steatosis was associated with aberrant microsomal apolipoprotein (apo) B-100 and microsomal triglyceride transfer protein (MTP) content, hypotriglyceridemia, hypocholesterolemia and abnormalities in both circulating lipoprotein composition and size. Atypical hepatobiliary sterol metabolism was established by the assessment of the activity of key intracellular enzymes for cholesterol homeostasis, which demonstrated, with respect to controls, a 40% reduction in 3-hydroxy-3-methylglutaryl coenzyme A reductase, a 30% reduction in cholesterol 7alpha-hydroxylase, and a 54% reduction in acyl CoA:cholesterol acyltransferase. During a 6-h biliary drainage, a decline in the bile acid output was recorded and might be linked to the low protein expression of the bile salt export pump (BSEP or ABCB11). Our data emphasize the crucial role of copper balance in hepatic sterol homeostasis and lipoprotein metabolism in LEC rats. Additional studies are needed to delineate the mechanisms of these disorders.     

Retrograde intrabiliary injection of amphipathic materials causes phospholipid secretion into bile. Taurocholate causes phosphatidylcholine secretion, 3-[(3-cholamidopropyl)dimethylammonio]-propane-1-sulphonate (CHAPS) causes mixed phospholipid secretion.

The control of biliary phospholipid and cholesterol secretions by bile acid was studied by using the technique of retrograde intrabiliary injection. Taurocholate (TC), a moderately hydrophobic bile acid, taurodehydrocholate (TDHC), a hydrophilic non-micelle-forming bile acid, and 3-[(3-cholamidopropyl)-dimethylammonio]propane-1-sulphonate (CHAPS), a detergent, were individually administered by retrograde intrabiliary injection (RII) into the biliary tree, and bile acids, phospholipids and cholesterol subsequently appearing in the bile were measured. TC (1.3 mumol; 45 microliters) injected retrogradely provoked a 3.5-fold increase in biliary phospholipid output for 40 min, as compared with the saline control. Injection of 2.7 mumol of TC (90 microliters) caused a 7.5-fold increase in phospholipid output, which reached a peak at 12 min after RII, and phospholipid output continued for 40 min. Cholesterol output was also elicited under these conditions, showing both dose-dependency and extended secretion. Injection of 1.8 mumol of TDHC caused very little increase in either biliary phospholipid or cholesterol. Injection of 0.9 mumol of CHAPS (45 microliters) provoked a single substantial peak of phospholipid output in the 3 min bile sample. T.l.c. analysis of the phospholipid extracts of the bile collected after each compound showed, for TC, a single compound which co-migrated with the phosphatidylcholine standard, whereas for CHAPS substantial amounts of other phospholipids were present.     

Reduction in plasma cholesterol and increase in biliary cholesterol by a diet rich in n-3 fatty acids in the rat

Cholesterol and lipoprotein metabolism were investigated in a group of rats fed a fish oil-supplemented diet, a rich source of n-3 fatty acids. For comparison purposes, other groups of rats were fed either safflower oil (n-6 fatty acids) or coconut oil (saturated fatty acids). Diets were isocaloric and contained identical amounts of cholesterol. Rats fed fish oils for 2 weeks showed a 35% lower plasma cholesterol level than rats fed safflower oil, who in turn showed a 14% lower plasma cholesterol level than those fed coconut oil. The fall in plasma cholesterol level with fish oils was associated with significant falls in low density and high density lipoprotein cholesterol levels, but with no significant change in the ratio of low density to high density lipoprotein cholesterol. The fatty acid compositions of plasma, hepatic, and biliary lipids showed relative enrichment with n-3 fatty acids, reflecting the composition of the diet. The fish oil diet increased the basal secretion rate of cholesterol into bile, but the bile acid secretion rate remained unchanged. It is suggested that n-3 fatty acids reduce the plasma cholesterol level in rats by increasing the transfer of cholesterol into bile.     

Biliary lipid secretion in the rat during infusion of increasing doses of unconjugated bile acids

The aim of the present study was to examine the secretion of biliary components in rats during infusion of increasing doses of either deoxycholic acid, chenodeoxycholic acid or cholic acid and to test the hypothesis that biliary phospholipids may regulate the hepatic bile acid secretory capacity. Analysis of bile samples, collected every 10 min throughout the infusion period showed that there was an elevation of bile acid, phospholipid, cholesterol and alkaline-phosphodiesterase secretion, with all the bile acids, peaking and then gradually declining. Their secretory rates maximum differed and were inversely related to their detergent strength. However, the secretory rates maximum and total output of phospholipids and cholesterol were similar for all bile acids infused. The per cent contribution of phosphatidylcholine to total bile acid-dependent phospholipid secretion was reduced from 84% (in the pre-infusion period) to 59, 46 and 13% at the end of the cholic acid, chenodeoxycholic acid and deoxycholic acid infusions, respectively. This decrease in the per cent contribution of phosphatidylcholine was associated with an increase in the contribution of both sphingomyelin and phosphatidylethanolamine. The biliary phospholipid fatty acid pattern corroborated these changes in the phospholipid classes. Since sphingomyelin and phosphatidylethanolamine are major phospholipids in bile canalicular and other hepatocellular membranes, the marked increase in their secretion in bile during the infusion of high doses of bile acids may indicate solubilization of membrane phospholipids, resulting in membrane structural changes responsible for the reduced excretory function of the liver.     

Hepatic metabolism and secretion of a cholesterol-enriched lipoprotein fraction

A potentially important source of cholesterol secreted in bile is cholesterol-rich lipoproteins. However, the fate of the cholesterol carried in these lipoproteins after hepatic uptake has not been investigated. We harvested an apoE- and cholesterol-rich lipoprotein fraction (d 1.02-1.06 g/ml) from hypercholesterolemic rats and examined the acute effects of these lipoproteins on hepatic cholesterol metabolism, very low density lipoprotein (VLDL) secretion, and biliary lipid secretion. Administration of a lipoprotein bolus (20 mg of cholesterol) to rats resulted in a significant decrease in 3-hydroxy-3-methylglutaryl coenzyme A reductase activity and a significant increase in acyl-coenzyme A:cholesterol acyltransferase activity over controls at 1 hr. Hepatic cholesteryl ester content increased 400% with no change in hepatic free cholesterol content or biliary cholesterol secretion. These cholesterol-rich lipoproteins delivered in the isolated perfused liver effected a fivefold increase in hepatic VLDL secretion with no change in composition. Therefore, cholesterol-rich lipoproteins do not acutely alter biliary cholesterol secretion. Rather, the majority of the cholesterol delivered to the liver in these lipoproteins is either esterified and stored as cholesteryl ester or resecreted as free and esterified cholesterol in hepatic VLDL.     

Biliary lipid secretion in the rat. The uncoupling of biliary cholesterol and phospholipid secretion from bile acid secretion by sulfated glycolithocholic acid

Glycolithocholic acid and its sulfated derivative are major metabolites of the secondary bile acid lithocholic acid in man. Both compounds are known to induce cholestasis in experimental animals. We compared the effects of these endogenous hepatotoxins on bile production and biliary lipid composition in rats with chronic biliary drainage. The compounds were administered enterally at relatively low rates (5-50% of the rats' endogenous bile acid secretion in these experiments) to simulate enterohepatic circulation. Both compounds were substantially secreted into bile (more than 90% of dose); sulfated glycolithocholic acid unchanged and glycolithocholic acid after hepatic hydroxylation predominantly in the form of glyco-beta-muricholic acid (cf. Kuipers et al. (1986) Am. J. Physiol. 251, G189-G194). Neither glycolithocholic acid nor its sulfated derivative affected the biliary excretion of endogenous bile acids or bile flow in these experiments. In spite of this, phospholipid and cholesterol secretion were significantly reduced by sulfated glycolithocholic acid but were not altered by glycolithocholic acid. Phospholipid and cholesterol secretion rapidly decreased to 25 and 50% of their initial values, respectively, at biliary output rates of sulfated glycolithocholic acid up to 2 mumol/h, and did not further decrease when this output was increased to 6 mumol/h. Small unilamellar liposomes consisting of cholesterol, [Me-14C]choline-labeled phosphatidylcholine, phosphatidylserine and [3H]cholesteryl oleate in a 5:4:1:0.1 molar ratio were employed to label intrahepatic lipid pools. Administration of sulfated glycolithocholic acid slightly reduced bile acid synthesis from [3H]cholesteryl oleate, but significantly reduced the biliary secretion of [14C]phospholipid. Glycolithocholic acid did not affect the hepatic processing of liposomal lipids. It is concluded that sulfated glycolithocholic acid at low doses causes the uncoupling of biliary lipid secretion from that of bile acids, which might represent in initiating event in sulfated glycolithocholic acid hepatotoxicity.     

The contribution of newly synthesized cholesterol to bile salt synthesis in rats quantified by mass isotopomer distribution analysis

A new stable isotope procedure has been developed and validated in rats, applying [1-(13)C]acetate infusion to quantify the production of bile salts from de novo synthesized cholesterol making use of the mass isotopomer distribution analysis (MIDA) principle. Ions (m/z) 458-461, 370-373 and 285-288 were monitored by GC/MS (EI-mode) for the methyl trimethylsilylether derivatives of cholate, chenodeoxycholate and beta-muricholate, respectively. Rats with intact exteriorized enterohepatic circulation and rats with chronic bile diversion were infused with [1-(13)C]acetate for up to 14 h. After 10 h of infusion the enterohepatic circulation of the intact group was interrupted to deplete the existing bile salt pool (acute bile diversion). The fractions of biliary cholesterol and individual bile salts derived from newly synthesized cholesterol were determined by MIDA at t=14 h. In rats with acute bile diversion, these fractions were 20, 25, 27 and 23% for biliary cholesterol, cholate, chenodeoxycholate and beta-muricholate, respectively. After bile diversion for 8 days to induce hepatic cholesterol and bile salt synthesis, these fractions increased significantly to 32, 47, 41 and 47%, respectively. Calculated enrichments of the acetyl-CoA precursor pools were similar for all bile salts and biliary cholesterol within the two rat groups. However, chronic enterohepatic interruption decreased the acetyl-CoA pool size almost two-fold. We conclude that MIDA is a validated new stable isotope technique for studying the synthetic pathway from acetyl-CoA to bile salts. This technique provides an important new tool for studying bile salt metabolism in humans using stable isotopes.     

Contraceptive steroids increase cholesterol in bile: mechanisms of action

Contraceptive steroids increase the risk of acquiring cholesterol gallstones. The factors responsible include an increase in cholesterol saturation of bile and an increase in rate of secretion of cholesterol into bile. The goal of this study was to investigate the mechanism(s) of these increases in biliary cholesterol. During the use of contraceptive steroids, cholesterol saturation of gallbladder bile and the amount of cholesterol secreted per mole of bile acid increased (P less than 0.05 and P less than 0.02, respectively). Cholesterol absorption, cholesterol synthesis, chylomicron remnant clearance, and the concentration of plasma and lipoprotein lipids were not altered by contraceptive steroids. Despite this apparent lack of effect, important correlations were present during steroid use. LDL (low density lipoprotein) cholesterol increased as dietary cholesterol increased (r = 0.58, P less than 0.025). Cholesterol synthesis correlated directly with VLDL cholesterol concentration (r = 0.64, P less than 0.01), biliary cholesterol secretion (r = 0.68, P less than 0.01) and with molar percent cholesterol in bile (r = 0.49, P = 0.06). Chylomicron remnant clearance also correlated with cholesterol secretion (r = 0.85, P less than 0.001). As either remnant uptake or synthesis increased, the effect of the other source of hepatic cholesterol on biliary cholesterol secretion diminished. These relationships were not observed in the same subjects when they were not taking the hormones. The findings suggest that both newly synthesized and dietary cholesterol contribute to the cholesterol secreted in bile. This is consistent with the hypothesis that cholesterol for secretion into bile and VLDL is derived from a common metabolic pool of free cholesterol. It is proposed that contraceptive steroids exert their effect on biliary cholesterol by increasing cholesterol entering the pool and/or by inhibiting hepatic ACAT (acylcoenzyme A:cholesterol acyltransferase) activity, a known effect of progesterone, so that an increase in free cholesterol entering the pool leads to an increase in output.